What is EGETM?
Biocytogen’s Extreme Genome Editing System (EGE) is a set of high efficiency gene knockout/knockin methods based on CRISPR/Cas9 technology and developed and optimized by Biocytogen (hereinafter referred to as the EGETM system).
What services can be provided by the EGETM system?
EGETM can be widely used for high-efficiency genome editing of various eukaryotic systems, including gene fixed-point knockouts, point mutations, knockin, etc. It can be used in rats, mice and various cell lines. Biocytogen has successfully prepared more than 200 types of knockouts, conditional knockout and knockin mouse models, more than 50 types of rat models and more than 70 types of fixed-point gene editing cell lines (human embryonic stem cells/ips cells and multiple types of cancer cell lines, etc.) by making use of EGETM.
Compared to standard CRISPR/Cas9 technology, what are the advantages of the EGETM system?
The use of CRISPR/Cas9 technology for gene spot mutation and knockin is based on homologous recombination of the genome; the homologous recombination efficiency of exogenous DNA mediated by common CRISPR/Cas9 technology is low. Through a series of innovation and optimization strategies, the homologous recombination efficiency for the EGETM system developed by Biocytogen is increased by 10-20 times when compared to traditional CRISPR/Cas9 technology, making gene modification with EGETM more efficient, faster, and convenient.
How to avoid off-target and random insertion issues when using CRISPR/Cas9technology? What are the differences between EGETM by Biocytogen and standard CRISPR/Cas9 technology in this regard?
It is widely known that CRISPR/Cas9 may have off-target problems. Although the off-target efficiency of CRISPR/Cas9 is deemed to be low in references (almost negligible), Biocytogen optimized this aspect even more for the EGETM system: when designing sgRNA and after a thorough literature search, off-target probability is used as the most important design metric, so as to ensure that the off-target efficiency is the lowest. This was shown in preliminary off-target experiments by Biocytogen. In addition, during preliminary design, only the sgRNA with possible off-target positions at obvious genome redundant sequences are selected; i.e. sgRNA that may be subject to off-target effects on functional genomic sequences are abandoned; all this ensures that EGETM will not cause off-target effects on your experimental result.
As for gene knockin by CRISPR/Cas9 technology, random insertion of exogenous DNA into the genome might occur; this is also a possibility when using Biocytogen’s EGETM system. Biocytogen provides Southern blot mouse tail detection for the conditional knockout/knockin mouse prepared by EGETM technology; Southern is the gold standard for verification of DNA random insertions; double Southern tests can guarantee the correct insertion point and ensure that there are no random insertion.
EGETM system is innovated and optimized based on CRISPR/Cas9 technology, then will such innovation and optimization cause impact on other functions of rats, mice and cell lines?
The goal behind the innovation and optimization of EGETM by Biocytogen is to improve the genetic recombination efficiency of traditional CRISPR/Cas9 technology; no chemical and physical material interference is adopted during this process, and there is no impact on the gene status and physiological status of the targeting subject (such as rat, mouse, human pluripotent stem cell, etc.). This ensures the smooth implementation of subsequent experiments.
Is it necessary to perform Southern test for CRISPR/Cas9 knockout mouse?
Southern blot is an important step during the preparation process of knockin and conditional knockout mouse models. Based on past experience, when the ES cell method is used for the preparation, the probability of random insertion is about 20%; if CRISPR/Cas9 is used, the probability of random insertion increases to 35%. Random insertions can only be eliminated by Southern blot detection. It is better if your service provider performs Southern blot detection, otherwise your time and money investment may go up in smoke.
How does the company screen for transfected ESCs? How to ensure the correct insertion of the target gene and karyotype change of long-term in vitro culture of ESCs?
During targeting vector construction, a positive selection marker (NEO resistance gene) and a DTA negative selection marker will be introduced; at the same time, a enzyme restriction site is introduced to a the specific position in the targeting vector. After electrotransformation of the targeting vector into the cell, resistance screening using G418 is performed first. Next, we perform Southern hybridization assays in triplicate to verify the two ends of the insertion point, so as to ensure that the target gene is inserted in the correct position, and that there are no random insertion on other sites. In addition, we monitor karyotype changes in ESCs subjected to long-term in vitro culture by performing karyotype analysis. Using cellular level chromosome banding technology, the chromosomes are analyzed, compared, sequenced and numbered according to chromosome length, centromere position and arm ratio, ensuring that subsequent micro-injection is performedonly after high-quality ESC is obtained.
What factors influence the targeting rate of target genes in embryonic stem cells? What is the positive rate in most circumstances?
The most important factor is the strain of the mouse embryonic stem cell; it is easy for some strains and difficult for others. Generally, the number reported in literature is 0.5-3%, but it is much easier for some loci, such as the ROSA26locus (which reach 10-40%). Generally, using the long homologous arm for the targeting vector improves the recombination efficiency. If DNA from C57BL/6 is used for targeting vector construction and recombination is performed with embryonic stem cells from 129, then the recombination efficiency may be affected; the efficiency will be relatively high if recombination is performed with embryonic stem cells from C57BL/6. Generally, Biocytogen uses two long homologous arms (each long homologous arm is between7-12kb). Meanwhile, the use of self-separated C57BL/6embryonic stem cells with high recombination rates increases the overall recombination rate by 10-30%.
What materials are requested from the customer to prepare knockout mice?
Customers only need to provide the name of gene to be used and any special requirements. We provide consulting services for free for inexperienced customers, in order to clearly define your needs and requirements and provide you the optimal design scheme. We also perform a sequence analysis for the gene, and then give you a preliminary design. After constant communication between us and the customer, and when consensus is reached between both parties, a technical service agreement is signed. We will then prepare your model until you ultimately receive your desired F1 mouse.